Currently there is a growing demand for cholesterol of pharmaceutical grade having a cholesterol content of 95% or more for the production of vitamins D2, D3, hormones and W/O emulsions in cosmetics.
Currently cholesterol at industrial scale is mainly produced from wool wax alcohols, i.e. the non-saponifiable fraction of wool grease, which contains from about 25% to about 32% of cholesterol. Most common processes for producing cholesterol comprise the formation of an insoluble addition product by reacting cholesterol with a metal salt followed by the decomposition of the adduct and the recovery of cholesterol. Such processes are able to meet the requirements of purity for pharmaceutical applications of cholesterol. For example, U.S. Pat. No. 2,536,753 discloses a process, wherein the metal salt is zinc chloride.
However, this known process generates large amounts of liquid industrial waste (LIW), the management of which can significantly increase production costs. In addition worldwide demand for wool has declined steeply over the past decades, which has led to far smaller sheep stocks and lower availability of wool grease, making it necessary to look at additional sources of supply of cholesterol.
Spiric A. et al: “Statistical evaluation of fatty acid profile and cholesterol content in fish (common carp) lipids obtained by different sample preparation procedures” Analytical Chimica Acta, vol. 672, no. 1-2 Jul. 2010, pp. 67-71 discloses a process for extracting cholesterol from fish tissues by saponification at 80° C. followed by extraction with hexane and diethyl ether.
GB 526951 discloses a process for the extraction of cholesterol from animal tissues such as brain, spinal cord, etc. by saponification and extraction with a non-water miscible solvent.
GB 489623 discloses a process for obtaining cholesterol from marine animal oils by subjecting the oil to fractionation by multiple sequential vacuum distillations of the oil at different temperatures and pressures, wherein one of the distillate fraction comprise cholesterol, both free and esterified. Such fraction comprising cholesterol, if desired, may be further purified by saponification of the fraction followed by extraction of non-saponifiable matter, concentration and crystallization.
In Example 1 of GB 489623, clarified whale oil is subjected to molecular distillation at a temperature of 90° C. to 220° C. and pressure of 0.001 to 0.003 mmHg. As the pressure is lowered and the temperature is raised, successive fractions amounting to 0.2 to 2% are withdrawn, such fractions comprising most of the free fatty acids, squalene and other volatiles. More fractions in proportions ranging from 0.5 to 10% are withdrawn between about 120° C. and 160° C., such fractions comprising free and esterified cholesterol. It is evident that that no less than four consecutive distillations, each at some specific temperature and pressure, are required to arrive at a cholesterol rich fraction using this process.
There are several other disadvantages of the process disclosed by GB 489623 as well. At present, fish oil is a valuable commodity due to its content of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid. Multiple distillations of fish oil increase the trans fatty acid content of the oil, and promote polymerization of unsaturated fatty acid, which in turn decreases the content of EPA and DHA. Multiple distillations thus render the fish oil unsuitable for human or animal consumption.
On the other hand, present day fish oils contain a great variety of toxic and/or harmful anthropogenic contaminants like polychlorinated biphenyls (PCB), dichlorodiphenyltrichloroethane (DDT) and its metabolites, dibenzo-dioxins (PCDDs), and dibenzo-furans (PCDFs), poly-aromatic hydrocarbons (PAH), pesticides and their degradation products, also known as persistent organic pollutants or POP's, which are resistant to environmental degradation and thus bio-accumulate. Therefore, the distillate fractions comprising cholesterol will comprise as well one or more of such contaminants. The content of such contaminants in the distillate fractions will be even higher than in the fish oil. This fact, though evident, can be found in the prior art.
U.S. Pat. No. 7,678,930 discloses a process for obtaining a free cholesterol-reduced fish oil by vacuum stripping the oil. On the other hand, U.S. Pat. No. 7,718,698 discloses a process for decreasing the amount of environmental pollutants in fish oil, also by vacuum stripping the oil. These two patents have similar disclosures. Therefore, under conditions of vacuum distillation where environmental pollutants are removed, free cholesterol is removed as well and vice versa.
The distillate of the process of U.S. Pat. No. 7,678,930 has a level of toxic and/or harmful anthropogenic contaminants higher than the fish oil and its cholesterol content is no greater than 10%, therefore it is unsuitable as a source of cholesterol in formulated shrimp and prawn feed. The same can be said of the cholesterol concentrates obtained by the process disclosed in GB 489623. Because cholesterol is obtained from such concentrates in GB 489623 by methods such as saponification followed by extraction of the non-saponifiable matter (which comprises all the POPs as well) with a water immiscible solvent, concentration and crystallization, the crystallized solid cholesterol will also contain contaminants, which by itself is sufficient to preclude its use for pharmaceutical purposes.
U.S. Pat. No. 4,104,286 discloses a process for isolating cholesterol from dried whole egg.
US 2011/0207952 discloses a process of cholesterol extraction from an algal processing waste discloses a process of saponifying a fat or oil, extracting with solvent the saponified mixture and extracting cholesterol from the solution stream.
JPS 63174997 with supercritical carbon dioxide.
International Application WO2016/096989 discloses a method for extracting cholesterol from a fish oil waste residue, the residue of a standard process for the production of concentrates of EPA and DHA from fish oil, containing up to 15% of cholesterol. It is known to a skilled person that such residue corresponds to about 1% of the original fish oil, which is equivalent to less than 10% of cholesterol present in original fish oil.